Cd137 as a proliferation factor for hematopoietic stem cells

ABSTRACT

The invention provides the CD137 molecule or a functional analogue thereof, for use in stimulating hematopoiesis. The invention further provides a method of treatment of a disorder characterized by insufficient numbers of cells of the hematopoietic system, including but not limited to T cells, B cells, granulocytes, macrophages, mesenchymal cells, osteoclasts and multipotent adult progenitor cells, comprising the application of CD137 or a functional analogue thereof.

FIELD OF THE INVENTION

The invention relates to the field of hematopoietic growth factors; inparticular, the invention relates to the induction of growth ofhematopoietic stem cells.

BACKGROUND OF THE INVENTION AND PRIOR ART

The cytokine receptor CD137 is a member of the tumour necrosis factorreceptor family. CD137 is expressed by activated T and B lymphocytes andexpression in primary cells is strictly activation dependent (Schwarz etal., 1995). Soluble forms of CD137 are generated by differentialsplicing and are found at enhanced concentrations in sera of patientswith rheumatoid arthritis (Michel et al., 1998). The gene for humanCD137 resides on chromosome 1p36, in a cluster of related genes, andthis chromosomal region is associated with mutations in severalmalignancies (Schwarz et al., 1997).

Crosslinking of CD137 costimulates proliferation of T lymphocytes(Goodwin et al., 1993; Pollock et al., 1993; Schwarz et al., 1996), andCD137 ligand expressed by B lymphocytes costimulates T cellproliferation synergistically with B7 (DeBenedette et al., 1995).

While agonistic antibodies and the ligand to CD137 enhance lymphocyteactivation, CD137 protein has the opposite effect. It inhibitsproliferation of activated T lymphocytes and induces programmed celldeath. These T cell-inhibitory activities of CD137 requireimmobilisation of the protein, arguing for transmission of a signalthrough the ligand/coreceptor (Schwarz et al., 1996). The known humanCD137 ligand is expressed constitutively by monocytes and its expressionis inducible in T lymphocytes (Alderson et al., 1994). Monocytes areactivated by immobilized CD137 protein and their survival is profoundlyprolonged by CD137. (Langstein et al., 1998; Langstein et al., 1999a).CD137 also induces proliferation in peripheral monocytes (Langstein etal., 1999b). Macrophage colony-stimulating factor (M-CSF) is essentialfor the proliferative and survival-enhancing activities of CD137(Langstein et al., 1999a; Langstein et al., 1999b).

WO99/44629, published Sep. 10, 1999, describes the use of CD137 inpromoting the proliferation of peripheral monocytes. The applicationteaches that CD137, in particular in a multimerized or immobilized form,may be used to enhance growth and proliferation of peripheral monocytes.The application teaches further that CD137 induces proliferation ofperipheral monocytes independently of hematopoietic stem cells (P. 4,line 25).

Cancer patients receiving a chemo- or/and radiation therapy suffer froma destruction or weakening of their immune and hematopoietic systems.The ability to reconstitute the immune and hematopoietic systems allowsthe application of higher doses of chemotherapy or/and radiation,increasing the chances of a complete removal of the tumor and metastases(Elias, 1995).

Sofar, granulocyte colony-stimulating factor (G-CSF) andgranulocyte/macrophage colony-stimulating factor (GM-CSF) are used forreconstituting the immune and hematopoietic systems (Fan et al., 1991;Neidhart, 1992; Hofstra et al., 1996; Sachs, 1996). These factors areknown to induce fever, chills, and other disagreeable and potentiallydangerous symptoms in patients receiving them.

However, the use of such prior art methods of stimulating hematopoiesisrests upon the use of mainly a single cytokine, namely, G-CSF. It isevident that the natural process of hematopoiesis is not driven by suchsingle factor alone, and hence, it is desirable to provide furtherfactor(s), which may be used to that end. Furthermore, the use of G-CSFis hampered by the relatively short half-life of that substance, so thatit is necessary to apply the factor repeatedly during the course oftreatment. This is associated with patient discomfort as a number ofintravenous applications are necessary. Still further, the response toG-CSF differs from patient to patient, and today it is not possible toforesee the response to the factor of a given patient. Therefore, in anumber of cases too little G-CSF is applied, or the hematopoietic systemdoes not rapidly enough or not to a large enough extent recover. Theconsequences of such insufficient response to the factor are anincreased number of treatment-associated complications, such asopportunistic infection. Such infections can not always be brought undercontrol, mainly because the patient's immune system is unable tosuccessfully cope with them in the first place. Therefore, a number ofpatients may be severely harmed as a consequence of such infection,while others die of the infection.

Thus, it is necessary to discover further factors, which are useful instimulating hematopoiesis by inducing proliferation and/ordifferentiation, in particular proliferation and/or differentiation ofbone marrow and/or peripheral hematopoietic stem cells. These factorsshould advantageously feature a long-lasting, stimulating effect on thehematopoietic system, so that a sufficient impact on stimulation and/orregeneration of the hematopoietic system can be achieved.

Hematopietic stem cells, mesenchymal cells and multipotent adultprogenitor cells contribute to the process of tissue repair andregeneration and wound healing (Jiang et al., 2002).

SUMMARY OF THE INVENTION

The invention solves these prior art problems by providing CD137molecule or a functional analogue thereof, for use in stimulatinghematopoiesis. The invention further provides a method of treatment of adisorder characterized by insufficient numbers of cells of thehematopoietic system, including but not limited to T cells, B cells,erythroid cells, granulocytes, macrophages, mesenchymal cells,osteoclasts and multipotent adult progenitor cells comprising theapplication to a mammal, including a patient, in need thereof of aneffective dose of CD137 or a functional analogue thereof.

The invention further comprises a method of treatment of a disordercharacterized by an insufficient number of cells of the hematopoieticsystem, including but not limited to T cells, B cells, erythroid cells,granulocytes, erythrocytes, macrophages, mesenchymal cells, osteoclastsand multipotent adult progenitor cells comprising the application ofCD137 or a functional analogue thereof to an isolated culture of stemcells or a mixed cell populations containing stem cells and the transferof the treated cells to a mammal, including a patient, in need thereof.

The invention also comprises a method for the treatment of a mammal inneed thereof wherein the mammal is characterized by having a decrease inthe number or activity of its white or red blood cells of normalphysiological function. Preferable, the decrease is caused by animmunodeficiency. Preferred immunodeficiencies include AIDS,hyperimmunoglobulin M syndrome, and other immunodeficiencies. Furtherpreferably, the decrease is caused by and/or associated with chemo-and/or radiotherapy. The chemo- and/or radiotherapy is preferablyadministered to treat cancer.

Also preferably, the decrease is associated with leukopenia. Theleukopenia is preferably caused by severe trauma, blood loss,immunodeficiency, or diseases such as agranulocytosis or bone marrowfailure. Bone marrow failure may be due to congenital factors, toxinssuch as benzene, drug abuse, viral infections such as hepatitis, or sideeffects of immunotherapies.

Further preferably, the decrease is associated with a disease such asanemia, aplastic anemia, megablastic anemia, myelophthisic anemia,myelodysplastic syndrome or hairy cell leukemia.

Also preferably, the decrease is associated with a decrease in red bloodcell number and/or function. The invention therefore provides a methodof treatment for a mammal in need thereof wherein the mammal suffersfrom a disease or condition characterized by a decrease in the numberand/or function of its red blood cells, comprising administering to themammal an effective amount of CD137 or of a functional analog thereof,or comprising contacting erythroid cells or erythroid precursor cellswith CD137 or a functional analog thereof, and administering theso-treated cells to the mammal. The cells may be derived from the mammalor from other sources. Preferably, they are derived from the mammal. Thedisorder or condition is preferably selected from anemia, anemiaassociated with renal failure or renal insufficiency, anemia ofprematurity, severe antepartum iron deficiency anemia, postpartumanemia, and pernicious anemia.

The dose of the CD137 protein or functional analogue thereof of theinvention is determined by the attending physician, or, in the case of anon-human mammal, by the researcher carrying out the experiment, or by aveterinarian. In general, effective doses of CD137 protein are withinthe range of 1 ng/kg to 1 mg/kg, more preferably 50 ng/kg to 100 μg/kg,most preferably 1 to 10 μg/kg.

The invention further provides a method for the stimulation of growth,proliferation, differentiation and/or activation of hematopoietic stemcells, comprising the step of contacting the cells with an effectiveamount of CD137, during a time period sufficient to allow for the saidstimulation of growth, proliferation, differentiation and/or activation.The amount of CD137 is preferably between 1 to 10 μg/kg. The time periodis preferably between 1 and 14 days.

The invention also provides the above method for the stimulation ofgrowth, proliferation and/or differentiation of cells that are moredifferentiated that stem cells, such as erythroid cells which may matureinto erythrocytes.

A further aspect of the invention is the provision of CD137 or afunctional analogue thereof, for use in induction of proliferation ofhematopoietic stem cells of a mammal. In another aspect, the inventionprovides CD137 or a functional analogue thereof, for use in stimulatinghematopoiesis. In a further aspect, the invention provides CD137 or afunctional analogue thereof, for use in tissue repair, tissueregeneration and wound healing. The invention also provides CD137 or afunctional analogue thereof, for use in enhancing innate and/or adaptiveimmunity for cancer therapy. Still further, the invention provides CD137or a functional analogue thereof, for use in enhancing innate and/oradaptive immunity for therapy of infectious disease. The invention alsoprovides CD137 or a functional analogue thereof, for use in enhancinginnate and/or adaptive immunity for vaccination against infectiousdisease. Also included in the scope of the invention is a method oftreatment of a disorder characterized by insufficient numbers of cellsof the hematopoietic system, including but not limited to T cells, Bcells, granulocytes, macrophages, mesenchymal cells, osteoclasts andmultipotent adult progenitor cells, comprising the step ofadministration to a mammal in need thereof of an effective dose of CD137or a functional analogue thereof. The invention further comprises amethod of treatment for a disorder characterized by an insufficientnumber of of cells of the hematopoietic system, including but notlimited to T cells, B cells, granulocytes, macrophages, mesenchymalcells, osteoclasts and multipotent adult progenitor cells comprising thestep of administration of CD137 or a functional analogue thereof to anisolated culture of stem cells and the transfer of the treated cells toa mammal in need thereof.

The invention also relates to a method for the treatment of a mammal inneed thereof wherein the mammal is characterized by having a decrease inthe number or activity of its white blood cells decrease is preferablycaused by or associated with an immunodeficiency. The immunodeficiencyis preferably selected from the group comprising AIDS,hyperimmunoglobulin M syndrome, radiation-induced immunodeficiency, andchemotherapy-induced immunodeficiency. The decrease is preferablyassociated with chemo- and/or radiotherapy and/or removal of bloodprogenitor cells. The chemo- and/or radiotherapy and/or removal of bloodprogenitor cells is preferably administered to treat cancer. The chemo-and/or radiotherapy and/or removal of blood progenitor cells is alsopreferably administered to treat autoimmune disease. Further preferably,the decrease is associated with leukopenia. The leukopenia is preferablycaused by a condition selected from the group comprising severe trauma,blood loss, immunodeficiency, or disease such as agranulocytosis or bonemarrow failure. The Bone marrow failure is preferably due to congenitalfactors, toxins such as benzene, street drugs, viral infections such ashepatitis, or side effects of immunotherapies. The leukopenia ispreferably caused by a disease such as anemia, aplastic anemia,megablastic anemia, myelophthisic anemia, myelodysplastic syndrome orhairy cell leukemia.

The dose of CD137 protein or functional analog thereof administered ispreferably within the range of 1 ng/kg to 1 mg/kg, more preferably 50ng/kg to 500 μg/kg. Further preferably, the dose of CD137 protein orfunctional analog thereof administered is within the range 100 ng/kg to100 μg/kg more preferably 500 ng/kg to 50 μg/kg. Still furtherpreferably, the dose of CD137 protein or functional analog thereofadministered is within the range of 1 to 10 μg/kg, more preferably 4 to6 μg/kg.

The invention also comprises a method for the stimulation of growth,proliferation, differentiation and/or activation of hematopoietic stemcells, comprising the step of contacting the cells with an effectiveamount of CD137 or a functional analog thereof, during a time periodsufficient to allow for said the stimulation of growth, proliferation,differentiation and/or activation. The concentration of CD137 ispreferably from about 1 ng/ml to about 1 mg/ml, more preferably fromabout 5 ng/ml to about 400 mg/ml, further preferably from about 15 ng/mlto about 100 ng/ml, most preferably about 60 ng/ml. Similarconcentration may be used with functional CD137 analogs, however. Theperson of skill in the art will appreciate that the concentrations ofsuch analog may differ from the actual CD137 concentrations give hereand that the optimum concentrations may be determined experimentally,for instance by determining the effective concentration of such analogsin functional assays such as the in vitro and in vivo assays describedin the examples hereinbelow.

Therefore, invention also comprises a method as described above whereinthe CD137 or functional analogue thereof is CD137, or a part of CD137,fused to Fc.

The CD137 used according to the invention preferably contains theextracellular part thereof. The CD137 preferably contains amino acids 18to 255 thereof. Further preferably, the CD137 contains amino acids 18 to186 thereof.

The invention also comprises the above methods wherein the CD137 orfunctional analogue thereof is used in combination with a growth factor.

The growth factor is preferably selected from among G-CSF, M-CSF,GM-CSF, IL-3, IFN-gamma, TNF, LIF, flt-3, and c-kit. More preferably,the growth factor is selected from among G-CSF, M-CSF and GM-CSF. Mostpreferably, the growth factor is G-CSF.

Preferably, in the methods of the invention, the CD137 or functionalanalogue thereof is administered as a single dose.

The invention also comprises a CD137 molecule, or functional analoguethereof, which is multimerized, for use according to the aboveembodiments or the above methods. The multimer preferably comprises 2 to20 monomers, more preferably 3 to 10 monomers, still more preferably 3to 5 monomers.

The monomers are preferably expressed a fusion protein. The monomers arealso preferably fused together by means of a covalent bond.

The invention also comprises a molecule capable of crosslinking CD137ligand(s) expressed on the surface of a target cell, for use accordingto the above embodiments of the invention.

The functional analog of CD137 is preferably an antibody or derived froman antibody. The functional analog is also preferably an anticalin orderived from an anticalin. Further preferably, the functional analog isa Trinectin or derived from a Trinectin. The functional analog ispreferably characterized by the ability to stimulate the growth,proliferation, differentiation and/or activation of stem cells. The stemcells are hematopoietic stem cells. The stem cells are furtherpreferably CD34 positive cells.

The invention also comprises a composition comprising CD137 or afunctional analogue thereof, and a diluent and/or carrier. Thecomposition of the invention is preferably suitable for dermal,transdermal, oral, intravenous, intraperitoneal, intramuscular, orintraliquoreal administration. The invention also provides a compositionof the invention for use in the treatment of a disorder of a mammalwherein the disorder is associated with decreased number or activity ofwhite blood cells. The decrease is preferably caused by or associatedwith an immunodeficiency. The immunodeficiency is preferably selectedfrom the group comprising AIDS, hyperimmunoglobulin M syndrome,radiation-induced immunodeficiency, chemotherapy-inducedimmunodeficiency.

Within the scope of the invention is included a composition as describedabove for use in stimulation of proliferation and/or differentiation ofhematopoietic stem cells. Such composition if preferably beneficial indisorders such as immunodeficiency, agranulocytosis, bone marrowfailure, anemia, aplastic anemia, megablastic anemia, myelophthisicanemia, myelodysplastic syndrome or hairy cell leukemia.

The invention also comprises a method of treatment of a mammal wherein atherapeutically effective amount of a composition of the invention isadministered to a mammal in need thereof, the mammal having a disorderassociated with decreased proliferation and/or differentiation of stemcells.

The disorder is preferably cancer of cells of the hematopoietic lineage.The disorder is further preferably leukemia.

The invention also provides CD137 or a functional analogue thereof, foruse in induction of proliferation of stem cells of a mammal. Also, Theinvention provides a method of treatment of a mammal in need thereof,wherein an effective dose of CD137 or a functional analog thereof isadministered to the mammal, wherein the mammal suffers from a disorderassociated with abnormal white blood cell number and/or function. Themammal preferably suffers from a disorder or condition selected fromimmunodeficiency, agranulocytosis, bone marrow failure, anemia, aplasticanemia, megablastic anemia, myelophthisic anemia, myelodysplasticsyndrome or hairy cell leukemia and the disorder or condition isimproved by the administration of CD137 or a functional analoguethereof.

The invention also provides inhibition of CD137 and/or inhibition ofCD137Ligand signalling, for use in the treatment of a disorder orcondition associated with excessive proliferation of hematopoieticcells.

The hematopoietic cells are preferably hematopoietic stem cells. Theinhibition is preferably mediated by antibodies to CD137 or toCD137Ligand. These antibodies should be inhibitory to the binding ofCD137 to CD137 Ligand, which may easily be determined by the skilledperson, for instance by means of a binding assay where one of thepartners is immobilized, for instance on a solid support such an anELISA plate, and the other partner is provided in a soluble, labeledform. The binding can then be detected by assaying the presence of thelabel on the solid support, and likewise, the inhibition of such bindingby the antibody may be ascertained by the absence or diminishing oflabel on the support in reactions where effective concentrations of suchantibody have been added. Of course, the antibody may be tested in otherways, for instance by determining its inhibitory effect on the functionof CD137 as measured in a functional assay as described hereinbelow inthe examples. The skilled person will be aware that such antibody may byitself have a functional, stem cell proliferative, growth-enhancing anddifferentiation-stimulating activity like the CD137 molecules andfunctional analog described herein. Sich activity may be determined forinstance in a functional assay as described in the examples hereinunder.Where such activity exists, and the antibody is to be used as inhibitorof the activation of cd137 Ligand through CD137, the preparation of Fabfragments or of single chain antibodies comprising a single bindingdomain only, will usually abolish such positive stimulatory activity, sothat the resulting Fab fragment of single chain antibody may then beused as a inhibitor. Similarly to inhibitory antibodies, other agentsmay be used that are capable of inhibiting CD137 Ligand or CD137expression. Such agents may be for instance antisense oligonucleotides,RNAi molecules, specific aptamers or aptazymes, which are capable ofreducing transcription and/or expression of CD137 or CD137 Ligand. Suchinhibition may then result in lowered stimulation of proliferationthrough the CD137/CD137 Ligand pathway, which in turn is desirable indisorders where cells of the hematopoietic lineage proliferate out ofcontrol. Such disorders include stem cell leukemia andmyeloproliferative disease.

Another use of the CD137 molecules of the invention is the provision ofthe coding sequence therefor, in particular, of the coding sequence ofthe extracellular domain thereof, preferably amino acids 18 to 255thereof, more preferably amino acids 18 to 186 thereof. This codingsequence is preferably provided in fused to transcriptional controlelements such as a promoter and optionally, an enhancer. The codingsequence is further preferably fused to a transcription terminatorand/or a Polyadenylation sequence. The promoter is preferably inducible,and also preferably tissue-specific. For instance, promoters specific towhite blood cells such as T cells, B cells, Monocytes, Granulocytes, andthe like are preferred. The coding sequence preferably comprises or isfused to a leader sequence, such as the leader sequence of CD137. Thecoding sequence may be provided as a multimer, such that one codingsequence is fused to at least one more coding seuqence. The codingseuqences are preferably not fised together directly, but sepoarated bya spacer sequence. The spacer sequence is preferably coding for one to50 amino acids, more preferably for 5 to 30, most preferably for 9 to 25amino acids. The spacer sequence may for instance be derived from thesequence coding for the hinge region of human antibodies. The codingsequence may thus be introduced into a cell which is then able toproduce and secrete the CD137 protein or fragment or multimer thereof.Thus, the so-produced CD137 fragment or multimer thereof may beadministered to a mammal in need thereof, according to the aboveembodiments of the invention. Alternatively, the cell comprising thecoding sequence may be introduced into the mammal, providing the mammalthough the CD137 produced by such cell internally with the Cd137molecules of the invention. Still further, the coding sequence may beintroduced into a mammal, either as naked DNA (for methods see e.g.,U.S. Pat. Nos. 5,580,859, 5,589,466, and 5,693,622), or through lipid-or liposome-mediated DNA uptake methods (see e.g., U.S. Pat. Nos.5,194,654, 5,223,263, 5,264,618, 5,459,127, and 5,703,055), or throughmethods where DNA uptake is mediated by other chemicals (see e.g., U.S.Pat. No. 6,022,874). Also methods using retroviral- or other biologicalmethods are well known to the person of skill in the art.

The CD137 protein as a soluble protein will generally be incative asconcerns the stimulation of proliferation, growth and/or differentiationof stem cells. Therefore, in order to achieve the acitvity of the CD137protein of the invention, it is generally necessary to multimerize theprotein, either by linking or fusing CD137 monomers, or by expressingthe CD137 protein on the surface of cells, or by introducing it toliposomes or the like bodies which are shaped similarly to cells, or byimmobilizing the CD137 protein on the surface of beads, or byimmobilizing it by using agents such as protein A, as describedhereinbelow. Similar considerations apply to functional analog of CD137,which may have to be multimerized as well in order to become functionalwithin the meaning of the invention.

Thus, the CD137 protein is preferably multimerized, preferably inmultimers of 2-20 molecules, more preferably in multimers of at least 3.Multimerization may be achieved by a variety of means. For instance,CD137 molecules may be coupled by chemical cross-linking. It isimportant not to destroy the biological activity of CD137 moleculescrosslinked in this manner. A number of cross-linkers have beendeveloped, some of which comprise spacer molecules in order to preventsteric hindrance to binding or other biological activities. Ascross-linker, the following molecules may be advantageously used:

Examples of homobifunctional crosslinkers include:

-   -   sulfosuccinimidyl        4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (sulfo-SMCC)        (see e.g., Z. Liu et al., J. Immunol. Methods 234(1-2): P153-67,        2000),    -   3,3′-dithio bis(sulfosuccinimidylpropionate),    -   BMME (Weston et al., Biochem Biophys. Acta 612, 40, 1980)    -   BSOCOES (Howard et al., J. Biol. Chem. 260, 10833, 1985),    -   DSP (Lee and Conrad, J. Immunol. 124, 518, 1985),    -   DSS D'Souza et al., J. Biol. Chem. 263, 3943, 1988),    -   EGS, optionally water-soluble (Geisler et al., Eur. J. Biochem.        206, 841, 1992, Moenner et al., PNAS 83, 5024, 1986, Yanagi et        al., Agric Biol. Chem. 53, 525, 1989),    -   SATA (Duncan et al., Anal. Biocehm. 132, 68, 1983).

Examples of heterobifunctional crosslinkers include:

-   -   GMBS (Kitagwa et al., J. Biochem. 94, 1160, 1983, Rusin et al.,    -   Biosens. Bioelectron. 7, 367, 1992),    -   MBS (Green et al., Cess 28, 477, 1982),    -   PMPI (Aithal et al., J. Immunol. Meth. 112, 63, 1988),    -   SMCC (Annunziato et al., Bioconjugate Chem. 4, 212, 1993),    -   SPDP (Caruelle et al., Anal. Biochem. 173, 328).

Crosslinkers are available commercially e.g., from Calbiochem, 10394Pacific Center Court, San Diego, Calif. 92121, USA, Pierce Chemicals4722 Bronze way, Dallas, Tex., 75236, USA, Dalton Chemical LaboratoriesInc. 349 Wildcat Rd., Toronto, ON, M3J 2S3, Canada.

Cross-linking of CD137 molecules may also be achieved through linkingthe molecules by using a peptide linker, or by attaching two or moreCD137 molecules to another protein. Such molecules may be expressedusing recombinant molecular DNA techniques. Another possibility iscoupling the CD137 molecule to a protein that is known to dimerize ormultimerize. Such proteins will, upon expression, dimerize ormultimerize, so that the CD137 moiety fused thereto will be dimerized ormultimerized as well. An example of this technology is the TNF-R-Fcfusion protein constructed by Peppel et al., J. Exp. Med. 174:1483-9,1991. A molecule constructed in that manner, Etanercept or Enbrel, isnow manufactured by Amgen and used for treatment of rheumatoidarthritis.

The biological activity of the cross-linked molecule obtained in thismanner may be assayed by any assay wherein the CD137 molecule or afunctional analogue thereof is tested for its ability to stimulate theproliferation, growth, differentiation and/or activation ofhematopoietic stem cells. Such assays may comprise stem cells,preferably peripheral stem cells, isolated from a mammal, preferably ahuman. The stem cells are preferably CD34 positive peripheral cells.Such cells may be isolated in ways known to the person of skill in theart, for instance, by panning on a surface covered with anti-CD34antibodies, by FACSort using labeled anti-CD34 antibodies, by isolationusing anti-CD34 antibodies bound to magnetic beads, or the liketechniques. An example of a test for stem cell proliferation isdescribed in example 1 hereunder.

The CD137 molecules of the invention preferably comprise the amino acidsequence of the native CD137 molecule, i.e., amino acids 18 to 255. Morepreferable, the CD137 molecule of the invention comprises the soluble,extracellular part of the CD137 molecule, i.e., amino acids 18 to 186.Of course, functional analogues of such molecules may be used as well.The transmembrane domain of CD137 (amino acids 18 to 186) mayadvantageously be included when it is desired to multimerize the CD137molecule through lipophilic affinities, for instance, in a liposome orwhen expressing the CD137 molecule on the surface of a cell.

Without wishing to be bound by theory, it is the inventor's belief thatthe effect of CD137 described herein is mediated by the cross-linking ofCD137 ligands on the cells that are so effected, preferably stem cellsTherefore, according to the invention any molecule may serve as afunctional analogue of CD137 within the context of this description,that is able to cross-link CD137 ligand(s) on target cells and in thatmanner causes stimulation of growth, proliferation, differentiationand/or activation of hematopoietic stem cells. Functional analogues ofCD137 may be found by deleting amino acid sequences from the sequence ofthe native CD137 that are not involved in binding CD137 ligand or whosedeletion does not interfere substantially with binding. The biologicaleffects of such molecules may be tested as mentioned hereinabove, inparticular as described in example 1. Further, it is possible toexchange amino acids within the sequence of CD137 with other aminoacids, preferably with similar amino acids, as described in more detailbelow. Still further, naturally occurring analogues and/or homologues ofCD137 may be used and tested for their activity on the desired mammalianstem cells which are preferably human. Such homologs include the murineCD137 homolog, 4-1BB (Kwon and Weissman, 1989). Further, anti-CD137Ligand antibodies, or fragments thereof having the ability to cross-linkCD137 molecules which are located on the surface of a cell membrane, maybe used in place of CD137. Also other members of the TNR-R family may beused, provided they (1) are able to cross-link CD137 molecules that arelocated on a cell surface membrane, or (2) they exert the actions ofCD137 described herein, that is, activation, proliferation,differentiation and/or growth.

The functional analogues of the CD137 molecule according to theinvention also comprise molecules that are not derived from CD137sequence. For instance, antibodies against CD137 ligand(s) may be used.The activity of such antibodies may be further enhanced by cross-linkingthe antibodies, e.g. using antibodies directed against the Fc part ofthe CD137 ligand(s)-binding antibodies. For instance, mouse antirabbit-antibodies could be used when the CD137 ligand(s) bindingantibody is a rabbit antibody.

Of course, the antigen-binding part of such antibodies may be used alone(e.g., as single-chain antibody) and multimerized in a like manner asdetailed above for CD137 protein. Multimerization of such antibodies maybe used to enhance the potency thereof.

Furthermore, the functional analogue may be selected from amongmolecules that, like antibodies, specifically bind to CD137 ligand(s)and, when such molecules are used in a multimerized form, are able tocross-link CD137 ligand. Such compounds can e.g. be antibodies, smallmolecules, recombinant phages, or peptides. Suitable molecules are e.g.,anticalins, described in EP1017814. Said European patent also describesthe process of preparing such anticalins with the ability to bind aspecific target. Further suitable molecules are Trinectins (Phylos Inc.,Lexington, Mass., USA, and Xu et al., Chem. Biol. 9:933, 2002). Anotherkind of suitable molecule are affybodies (see Hansson et al.,Immunotechnology 4(3-4):237-52, 1999, and Henning et al., Hum Gene Ther.13(12):1427-39, 2002, and references therein) The activity of CD137,fragments thereof, mutations thereof, cross-linked variants thereof, orgenerally of functional analogues thereof and of any variations andchanges to these molecules, within the context of the invention, may beascertained by the skilled person by assaying for (1) binding of suchanalogues, fragments, variants, etc. to CD137 ligand(s), or (2) byassaying the biological function of CD137 as described hereinabove,namely, the stimulation of growth, proliferation, differentiation and/oractivation of stem cells, preferably human or murine stem cells, morepreferably hematopoietic stem cells. Assays for the detection andmeasurement of growth, proliferation, differentiation or activation arewell known to the skilled person. For instance, growth may simply bedetermined by counting the cell number, subjecting the cells to theconditions of the assay, for instance by incubating the cells in thepresence and in the absence of a molecule of the invention, andrecounting the number of cells after a suitable period of time, whichwill generally be 1-14 days, preferably 2-10 days. Further by way ofillustration, the procedures described in the examples, for instance, inexample 1, and in examples 2 and 3, hereinbelow, may be used to assaycell proliferation. Activation of stem cells usually is accompanied byactivation of extracellular stress-regulated kinase (ERK1/2), ribosomalS6 kinase (p90RSK), Akt and the mitogen activated protein kinase (MAPK)pathway (see e.g., Lee et al., Blood. 99(12):4307-17, 2002;Rozenfeld-Granot et al., Exp Hematol. 30(5):473-80, 2002). Thus, theskilled artisan may determine for each of the molecules of the inventionwhether it is capable of activating, stimulating growth and/orstimulating proliferation, and/or stimulating differentiation of stemcells, by using hematopoietic stem cells or other stem cells, derivedfrom peripheral cells or from bone marrow. The stem cells may be humanor other stem cells. Preferably are murine or human stem cells. Furtherpreferred are bone marrow derived stem cells. Also preferred arehematopoietic stem cells. Most preferred are human bone-marrow derivedhematopoietic stem cells. Also preferred are human peripheralhematopoietic stem cells. CD34 positive cells are the preferred sourceof stem cells.

The CD137 molecules and functional analogues thereof which arecontemplated within the scope of the invention are useful for thestimulation of growth, proliferation, differentiation and/or activationof stem cells. Thus, these molecules are useful in the treatment ofdisorder where such stem cells lack activation, or where proliferationor where differentiation and/or growth thereof is beneficial. Suchdisorders and conditions include immunodeficiencies, including thosethat are associated with treatment with radiation therapy and/orchemotherapy. As such, the molecules of the invention may not only beadministered to humans, but also to other mammals, including but notlimited to dogs, cats, guinea pigs, hamsters, horses, rabbits, goats,sheep, cows, oxen, bulls, pigs, mice and rats.

Further, the molecules of the invention may be useful in enhancing thenumber of stem cells in poultry, including chicken and turkey.

The molecules of the invention may be used ex vivo, in order tostimulate growth, activation, differentiation and/or proliferation ofstem cells in a culture of such cells, or in vivo. For in vivo use,compositions need to be prepared that are compatible with theiradministration to a patient or non-human animal. Compositions that areinjected usually comprise the active ingredient together with apharmaceutically acceptable buffer and/or diluent. For dermalapplication, the active ingredient, which is usually a protein orpeptide, may be modified using the technique described by Foldvari etal., U.S. Pat. No. 6,444,200, included herein by reference. According tosaid Foldvari et al., a protein or peptide may be coupled to at leastone unsaturated fatty acid moiety having between 16-20 carbon atoms,which is covalently attached to the protein or peptide. The said fattyacid is preferably oleic acid. Further to enhance penetration of dermis,the methods described further hereinbelow for conferring on peptides theability to cross membranes, may also be used. Pharmaceuticalcompositions and preparations may comprise pharmaceutically acceptablecarriers and/or diluents. Pharmaceutically acceptable carriers ordiluents include those used in formulations suitable for oral orparenteral (e.g. intramuscular or intravenous) administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product. For example, formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the intended recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents and thickening agents,and liposomes or other microparticulate systems which are designed totarget the polypeptide to blood components or one or more organs.Suitable liposomes include, for example, those comprising the positivelycharged lipid (N[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium(DOTMA), those comprising dioleoylphosphatidylethanolamine (DOPE), andthose comprising3beta[N-(n′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol).Optionally, the proteins, peptides and/or other molecules of theinvention may be attached to drug delivery substrates in order tofacilitate their delivery to the correct location in the mammal,preferably the patient. Suitable substrates include polymeric beadsknown in the art. Such beads may, preferably, be engineered to targetthe peptides of the invention to a given location in the body.

The present invention also concerns the DNA sequence encoding a CD137protein and the CD137 proteins encoded by the DNA sequences. Such DNAsequences may be used according to the invention to express proteins orpeptides having the function of the CD137 proteins of the invention,i.e., stimulation of growth, proliferation, differentiation and/oractivation of hematopoietic stem cells.

Moreover, as mentioned hereinabove, the present invention furtherconcerns the DNA sequences encoding biologically active analogs,fragments and derivatives of the CD137 protein, and the analogs,fragments and derivatives encoded thereby. The preparation of suchanalogs, fragments and derivatives is by standard procedure (see forexample, Sambrook et al., 1989) in which in the DNA sequences encodingthe CD137 protein, one or more codons may be deleted, added orsubstituted by another, to yield analogs having at least one amino acidresidue change with respect to the native protein.

Of the above DNA sequences of the invention which encode a CD137protein, isoform, analog, fragment or derivative, there is alsoincluded, as an embodiment of the invention, DNA sequences capable ofhybridizing with a cDNA sequence derived from the coding region of anative CD137 protein, in which such hybridization is performed undermoderately stringent conditions, or preferably, under more stringent, ormost preferably, under stringent conditions, and which hybridizable DNAsequences encode a biologically active CD137 protein. These hybridizableDNA sequences therefore include DNA sequences which have a relativelyhigh homology to the native CD137 cDNA sequence and as such representCD137-like sequences which may be, for example, naturally-derivedsequences encoding the various CD137 isoforms, or naturally-occurringsequences encoding proteins belonging to a group of CD137-like sequencesencoding a protein having the activity of CD137. Further, thesesequences may also, for example, include non-naturally occurring,synthetically produced sequences, that are similar to the native CD137cDNA sequence but incorporate a number of desired modifications. Suchsynthetic sequences therefore include all of the possible sequencesencoding analogs, fragments and derivatives of CD137, all of which havethe activity of CD137.

To obtain the various above noted naturally occurring CD137-likesequences, standard procedures of screening and isolation ofnaturally-derived DNA or RNA samples from various cell types of mammals,preferably cells derived from hematopoietic precursors or suchprecursors, may be employed using the natural CD137 cDNA or portionthereof as probe (see for example standard procedures set forth inSambrook et al., 1989).

Likewise, to prepare the above noted various synthetic CD137-likesequences encoding analogs, fragments or derivatives of CD137, a numberof standard procedures may be used as are detailed herein belowconcerning the preparation of such analogs, fragments and derivatives.

A polypeptide or protein “substantially corresponding” to CD137 proteinincludes not only CD137 protein but also polypeptides or proteins thatare analogs of CD137.

Analogs that substantially correspond to CD137 protein are thosepolypeptides in which one or more amino acid of the CD137 protein'samino acid sequence has been replaced with another amino acid, deletedand/or inserted, provided that the resulting protein exhibitssubstantially the same or higher biological activity as the CD137protein to which it corresponds.

In order to substantially correspond to CD137 protein, the changes inthe sequence of CD137 proteins, such as isoforms are generallyrelatively minor. Although the number of changes may be more than fifty,they are preferably no more than about thirty, still more preferablyabout ten, more preferably no more than five, and most preferably nomore than three such changes. While any technique can be used to findpotentially biologically active proteins, which substantially correspondto CD137 proteins, one such technique is the use of conventionalmutagenesis techniques on the DNA encoding the protein, resulting in afew modifications. The proteins expressed by such clones can then bescreened for their activity in inducing stem cell proliferation, growth,differentiation and/or activation, for instance by using the assaysdescribed hereinbelow, e.g., in example 1, and in Examples 2 and 3hereinbelow.

“Conservative” changes are those changes which would not be expected tochange the activity of the protein and are usually the first to bescreened as these would not be expected to substantially change thesize, charge or configuration of the protein and thus would not beexpected to change the biological properties thereof.

Conservative substitutions of CD137 proteins include an analog whereinat least one amino acid residue in the polypeptide has beenconservatively replaced by a different amino acid. Such substitutionspreferably are made in accordance with the following list as presentedin Table A, which substitutions may be determined by routineexperimentation to provide modified structural and functional propertiesof a synthesized polypeptide molecule while maintaining the biologicalactivity characteristic of CD137 protein. TABLE A Original ExemplaryResidue Substitution Ala Gly; Ser Arg Lys Asn Gln; His Asp Glu Cys SerGln Asn Glu Asp Gly Ala; Pro His Asn; Gln Ile Leu; Val Leu Ile; Val LysArg; Gln; Glu Met Leu; Tyr; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser TrpTyr Tyr Trp; Phe Val Ile; Leu

Alternatively, another group of substitutions of CD137 protein are thosein which at least one amino acid residue in the polypeptide has beenremoved and a different residue inserted in its place according to thefollowing Table B. The types of substitutions which may be made in thepolypeptide may be based on analysis of the frequencies of amino acidchanges between a homologous protein of different species, such as thosepresented in Table 1-2 of Schulz et al., G. E., Principles of ProteinStructure Springer-Verlag, New York, N.Y., 1798, and FIGS. 3-9 ofCreighton, T. E., Proteins: Structure and Molecular Properties, W.H.Freeman & Co., San Francisco, Calif. 1983. Based on such an analysis,alternative conservative substitutions are defined herein as exchangeswithin one of the following five groups: TABLE B 1. Small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly); 2. Polarnegatively charged residues and their amides: Asp, Asn, Glu, Gln; 3.Polar, positively charged residues: His, Arg, Lys; 4. Large aliphaticnonpolar residues: Met, Leu, Ile, Val (Cys); and 5. Large aromaticresidues: Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles inprotein architecture. Gly is the only residue lacking any side chain andthus imparts flexibility to the chain. This however tends to promote theformation of secondary structure other than a-helical. Pro, because ofits unusual geometry, tightly constrains the chain and generally tendsto promote β-turn-like structures, although in some cases Cys can becapable of participating in disulfide bond formation which is importantin protein folding. Note that Schulz et al., supra, would merge Groups 1and 2, above. Note also that Tyr, because of its hydrogen bondingpotential, has significant kinship with Ser, and Thr, etc.

Conservative amino acid substitutions according to the presentinvention, e.g., as presented above, are known in the art and would beexpected to maintain biological and structural properties of thepolypeptide after amino acid substitution. Most deletions andsubstitutions according to the present invention are those which do notproduce radical changes in the characteristics of the protein orpolypeptide molecule. “Characteristics” is defined in a non-inclusivemanner to define both changes in secondary structure, e.g. α-helix orβ-sheet, as well as changes in biological activity, e.g., stimulation ofgrowth, proliferation, differentiation and/or activation of stem cells,and/or cross-linking of CD137 ligand(s), preferably CD137 ligand(s)expressed on stem cells.

Examples of production of amino acid substitutions in proteins which canbe used for obtaining analogs of CD137 proteins for use in the presentinvention include any known method steps, such as presented in U.S.patent RE 33,653, 4,959,314, 4,588,585 and 4,737,462, to Mark et al.;5,116,943 to Koths et al., 4,965,195 to Namen et al.; 4,879,111 to Chonget al.; and 5,017,691 to Lee et al.; and lysine substituted proteinspresented in U.S. Pat. No. 4,904,584 (Shaw et al.).

Besides conservative substitutions discussed above which would notsignificantly change the activity of CD137 protein, either conservativesubstitutions or less conservative and more random changes, which leadto an increase in biological activity of the analogs of CD137 proteins,are intended to be within the scope of the invention.

When the exact effect of the substitution or deletion is to beconfirmed, one skilled in the art will appreciate that the effect of thesubstitution(s), deletion(s), etc., will be evaluated by routinebinding, proliferation, growth, differentiation and/or activationassays, preferably using hematopoietic stem cells as target cells.Screening using such a standard test does not involve undueexperimentation.

Acceptable CD137 analogs are those which retain at least the capabilityof binding to CD137 ligand(s), and thereby, as noted above when used asin a multimerized form, are able to cross-link said CD137 ligand(s).

At the genetic level, these analogs are generally prepared bysite-directed mutagenesis of nucleotides in the DNA encoding the CD137protein, thereby producing DNA encoding the analog, and thereaftersynthesizing the DNA and expressing the polypeptide in recombinant cellculture. The analogs typically exhibit the same or increased qualitativebiological activity as the naturally occurring protein, Ausubel et al.,Current Protocols in Molecular Biology, Greene Publications and WileyInterscience, New York, N.Y., 1987-1995; Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989.

Preparation of a CD137 protein in accordance herewith, or an alternativenucleotide sequence encoding the same polypeptide but differing from thenatural sequence due to changes permitted by the known degeneracy of thegenetic code, can be achieved by site-specific mutagenesis of DNA thatencodes an earlier prepared analog or a native version of a CD137protein. Site-specific mutagenesis allows the production of analogsthrough the use of specific oligonucleotide sequences that encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 complementingnucleotides on each side of the sequence being altered. In general, thetechnique of site-specific mutagenesis is well known in the art, asexemplified by publications such as Adelman et al., DNA 2:183 (1983), orXu et al., Biotechniques 32:1266-8, 2002 (and references therein), thedisclosure of which is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily available commercially and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol. 153:3, 1987) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevantpolypeptide. An oligonucleotide primer bearing the desired mutatedsequence is prepared synthetically by automated DNA/oligonucleotidesynthesis. This primer is then annealed with the single-strandedprotein-sequence-encoding vector, and subjected to DNA-polymerizingenzymes such as E. coli polymerase I Klenow fragment, to complete thesynthesis of the mutation-bearing strand. Thus, a mutated sequence andthe second strand bear the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli JM101 or XL-1Blue cells, and clones are selected that include recombinant vectorsbearing the mutated sequence arrangement.

After such a clone is selected, the mutated CD137 protein sequence maybe removed and placed in an appropriate vector, generally a transfer orexpression vector of the type that may be employed for transfection ofan appropriate host.

Accordingly, gene or nucleic acid encoding for a CD137 protein can alsobe detected, obtained and/or modified, in vitro, in situ and/or in vivo,by the use of known DNA or RNA amplification techniques, such as PCR andchemical oligonucleotide synthesis. PCR allows for the amplification ofspecific DNA sequences by repeated DNA polymerase reactions. Thisreaction can be used as a replacement for cloning; all that is requiredis a knowledge of the nucleic acid sequence. In order to carry out PCR,primers are designed which are complementary to the sequence ofinterest. The primers are then generated by automated DNA synthesis.Because primers can be designed to hybridize to any part of the gene,conditions can be created such that mismatches in complementary basepairing can be tolerated. Amplification of these mismatched regions canlead to the synthesis of a mutagenized product resulting in thegeneration of a peptide with new properties (i.e., site directedmutagenesis). See also, e.g., Ausubel, supra, Ch. 16. Also, by couplingcomplementary DNA (cDNA) synthesis, using reverse transcriptase, withPCR, RNA can be used as the starting material for the synthesis of theCD137, or of a preferably functional part thereof, without cloning.

Furthermore, PCR primers can be designed to incorporate new restrictionsites or other features such as termination codons at the ends of thegene segment to be amplified. This placement of restriction sites at the5′ and 3′ ends of the amplified gene sequence allows for gene segmentsencoding CD137 protein or a fragment thereof to be custom designed forligation other sequences and/or cloning sites in vectors.

PCR and other methods of amplification of RNA and/or DNA are well knownin the art and can be used according to the present invention withoutundue experimentation, based on the teaching and guidance presentedherein. Known methods of DNA or RNA amplification include, but are notlimited to polymerase chain reaction (PCR) and related amplificationprocesses (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159,4,965,188, to Mullis et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 toTabor et al.; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464to Wilson et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No.5,066,584 to Gyllensten et al.; U.S. Pat. No. 4,889,818 to Gelfand etal.; U.S. Pat. No. 4,994,370 to Silver et al.; U.S. Pat. No. 4,766,067to Biswas; U.S. Pat. No. 4,656,134 to Ringold; and Innis et al., eds.,PCR Protocols: A Guide to Method and Applications) and RNA mediatedamplification which uses anti-sense RNA to the target sequence as atemplate for double stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek et al., with the tradename NASBA); and immuno-PCR which combinesthe use of DNA amplification with antibody labeling (Ruzicka et al.,Science 260:487 (1993); Sano et al., Science 258:120 (1992); Sano etal., Biotechniques 9:1378 (1991)), the entire contents of which patentsand reference are entirely incorporated herein by reference.

Similarly, derivatives may be prepared by standard modifications of theside groups of one or more amino acid residues of the CD137 protein, itsanalogs or fragments, or by conjugation of the CD137 protein, itsanalogs or fragments, to another molecule e.g. an antibody, enzyme,receptor, etc., as are well known in the art. Accordingly, “derivatives”as used herein covers derivatives which may be prepared from thefunctional groups which occur as side chains on the residues or the N-or C-terminal groups, by means known in the art, and are included in theinvention. Derivatives may have chemical moieties such as carbohydrateor phosphate residues, provided such a fraction has the same or higherbiological activity as CD137 proteins.

For example, derivatives may include aliphatic esters of the carboxylgroups, amides of the carboxyl groups by reaction with ammonia or withprimary or secondary amines, N-acyl derivatives or free amino groups ofthe amino acid residues formed with acyl moieties (e.g., alkanoyl orcarbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group(for example that of seryl or threonyl residues) formed with acylmoieties.

The term “derivatives” is intended to include only those derivativesthat do not change one amino acid to another of the twenty commonlyoccurring natural amino acids.

CD137 is a protein or polypeptide, i.e. a sequence of amino acidresidues. A polypeptide consisting of a larger sequence which includesthe entire sequence of a CD137 protein, in accordance with thedefinitions herein, is intended to be included within the scope of sucha polypeptide as long as the additions do not affect the basic and novelcharacteristics of the invention, i.e., if they either retain orincrease the biological activity of CD137 protein or can be cleaved toleave a protein or polypeptide having the biological activity of CD137protein. Thus, for example, the present invention is intended to includefusion proteins of CD137 protein or part of the CD137 protein with otheramino acids or peptides, for instance as described hereinabove.

The CD137 protein, their analogs, fragments and derivatives thereof,have a number of possible uses, for example:

CD137 protein, its analogs, fragments and derivatives thereof, may beused to enhance the function of naturally-occurring CD137 in mammals,specifically, in stem cells and/or in cells of the hematopoietic systemand/or lineage. For example, if CD137 is expressed on the surface of acell, which comes in contact with stem cells, then introducing the CD137gene into such cells of a mammal can enhance hematopoiesis bystimulating the proliferation, growth, differentiation and/or activationof stem cells. In this case the CD137 protein, its analogs, fragments orderivatives thereof, which have the desired stem cell stimulatingeffect, may be introduced to the cells by standard procedures known perse. It is possible to introduce the CD137 gene as described furtherabove, or as illustrated in the examples below. Another possibility isto introduce the sequences of the CD137 protein (e.g., any one of theCD137 or its isoforms) in the form of oligonucleotides which can beabsorbed by the cells and expressed therein.

Another possibility is to use antibodies specific for the CD137 proteinto inhibit its stem cell stimulating effects. This may be desirablewhere the immune system of a patient is overly active, e.g., in cases ofautoimmune diseases such as arteriosclerosis, arthritis, Crohn'sdisease, Hashimoto's thyroiditis, Addison's disease, juvenile diabetes,diabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, dermatomyositis, lupuserythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome,Graves disease, systemic lupus erythematosus, ulcerative colitis,psoriasis, multiple sclerosis, myasthenia gravis, and other diseasescaused by or associated with autoimmunity, or in cancers arising fromthe hematopoietic system, such as granulomas, myeloproliferativedisease, stem cell leukemia, other leukemias and generally all types ofmalignant growth associated with excess growth of cells derived from theheamtopoietic system.

One way of inhibiting the CD137 protein is by a ribozyme approach.Ribozymes are catalytic RNA molecules that specifically cleave RNAs.Ribozymes may be engineered to cleave target RNAs of choice, e.g., themRNAs encoding the CD137 protein of the invention. Such ribozymes wouldhave a sequence specific for the CD137 protein mRNA and would be capableof interacting therewith (complementary binding) followed by cleavage ofthe mRNA, resulting in a decrease (or complete loss) in the expressionof the CD137 protein, the level of decreased expression being dependentupon the level of ribozyme expression in the target cell. To introduceribozymes into the cells of choice, any suitable vector may be used,e.g., plasmid, bacterial vectors, that are usually used for this purpose(see also (i) above, where the vector has, as second sequence, a cDNAencoding the ribozyme sequence of choice). (For reviews, methods etc.concerning ribozymes see Chen et al., 1992; Zhao and Pick, 1993; Shoreet al., 1993; Joseph and Burke, 1993; Shimayama et al., 1993; Cantor etal., 1993; Barinaga, 1993; Crisell et al., 1993 and Koizumi et al.,1993).

Further, CD137 expression may be inhibited by using antisenseoligodeoxynucleotides and/or RNA interference through small interferingRNAs or small hairpin RNAs (see e.g., Hannon, Nature 418(6894):244-51,2002, Ueda, J. Neurogenet. 15(3-4):193-204, 2001, Lindenbach and Rice,Mol. Cell. 9(5):925-7, 2002, Brantl, Biochim Biophys Acta.1575(1-3):15-25, 2002.

As noted hereinabove and hereinbelow, the CD137 protein, or its analogs,fragments or derivatives thereof, of the invention may also be used asimmunogens (antigens) to produce specific antibodies thereto. Theseantibodies may also be used for the purposes of purification of theCD137 protein (e.g., CD137 or any of its isoforms) either from cellextracts or from transformed cell lines producing CD137 protein, or itsanalogs or fragments.

It should also be noted that the isolation, identification andcharacterization of the CD137 protein of the invention may be performedusing any of the well known standard screening procedures. As notedabove and below, procedures may be employed such as affinitychromatography, phage display, DNA hybridization procedures, etc. as arewell known in the art, to isolate, identify and characterize the CD137protein of the invention or to isolate, identify and characterizeadditional proteins, factors, etc. which are capable of binding to theCD137 ligand and/or to stimulate the proliferation, growth,differentiation and/or activation of stem cells.

As set forth hereinabove, the CD137 protein may be used to generateantibodies specific to CD137 proteins, e.g., CD137 and its isoforms.These antibodies or fragments thereof may be used as set forthhereinbelow in detail, it being understood that in these applicationsthe antibodies or fragments thereof are those specific for CD137proteins.

Since it may be advantageous to design peptide inhibitors thatselectively inhibit CD137 activity without interfering with otherphysiological processes in which other proteins are involved, the poolof peptides binding to CD137 in an assay such as the one described byGeysen (Geysen, 1985; Geysen et al., 1987) can be further synthesized asa fluorogenic peptide to test for selective binding to such otherproteins to select only those specific for CD137 Such peptides may thenfurther investigated for inhibition of the binding of CD137 to itsligand(s). Peptides which are able to inhibit such interaction may thenbe selected as candidate structures for molecules that are able toinhibit the activity of CD137 described hereinbelow and hereinabove.Peptides which are determined to be specific can then be modified toenhance cell permeability and inhibit the activity of CD137 eitherreversibly or irreversibly. Accordingly, peptides that selectively bindto CD137 can be modified with, for example, an aldehyde group,chloromethylketone, (acyloxy)methyl ketone or a CH₂OC (O)-DCB group tocreate a peptide inhibitor of CD137 activity. Further, to improvepermeability, peptides can be, for example, chemically modified orderivatized to enhance their permeability across the cell membrane andfacilitate the transport of such peptides through the membrane and intothe cytoplasm. Muranishi et al. (1991) reported derivatizingthyrotropin-releasing hormone with lauric acid to form a lipophiliclauroyl derivative with good penetration characteristics across cellmembranes. Zacharia et al. (1991) also reported the oxidation ofmethionine to sulfoxide and the replacement of the peptide bond with itsketomethylene isoester (COCH₂) to facilitate transport of peptidesthrough the cell membrane. Hildt and Schmidt (EP1127133) describe apeptide derived from Hepatitis Virus B protein which is capable ofmediating cell permeability of a protein, peptide, or DNA it is mixedwith or coupled to. These are just some of the known modifications andderivatives that are well within the skill of those in the art.

The methods of mediating cell permeability for a protein, peptide ornucleic acid molecule described hereinabove and hereinbelow may ofcourse be used in the preparation of the antisense oligonucleotides andinterfering RNA molecules of the invention that are describedhereinabove. The ability of such molecules to cross membranes will ofcourse enhance their ability to reach their target which is typicallylocated inside of a cell.

U.S. Pat. No. 5,149,782 discloses conjugating a molecule to betransported across the cell membrane with a membrane blending agent suchas fusogenic polypeptides, ion-channel forming polypeptides, othermembrane polypeptides, and long chain fatty acids, e.g. myristic acid,palmitic acid. These membrane blending agents insert the molecularconjugates into the lipid bilayer of cellular membranes and facilitatetheir entry into the cytoplasm.

Low et al., U.S. Pat. No. 5,108,921, reviews available methods fortransmembrane delivery of molecules such as, but not limited to,proteins and nucleic acids by the mechanism of receptor mediatedendocytotic activity. These receptor systems include those recognizinggalactose, mannose, mannose 6-phosphate, transferrin,asialoglycoprotein, transcobalamin (vitamin B₁₂), α-2 macroglobulins,insulin and other peptide growth factors such as epidermal growth factor(EGF). Low et al. teaches that nutrient receptors, such as receptors forbiotin and folate, can be advantageously used to enhance transportacross the cell membrane due to the location and multiplicity of biotinand folate receptors on the membrane surfaces of most cells and theassociated receptor mediated transmembrane transport processes. Thus, acomplex formed between a compound to be delivered into the cytoplasm anda ligand, such as biotin or folate, is contacted with a cell membranebearing biotin or folate receptors to initiate the receptor mediatedtrans-membrane transport mechanism and thereby permit entry of thedesired compound into the cell.

Methods for introducing proteins into cells are described, e.g., inEP1127133 and WO0026379, which publications are included herein in theirentirety by reference.

As will be appreciated by those of skill in the art of peptides, thepeptide inhibitors of the CD137 interaction according to the presentinvention is meant to include peptidomimetic drugs or inhibitors, whichcan also be rapidly screened for binding to CD137 to design perhaps morestable inhibitors.

The methods of mediating cell permeability for a protein, peptide ornucleic acid molecule described hereinabove may of course be used in thepreparation of the antisense oligonucleotides and interfering RNAmolecules of the invention that are described hereinabove. The abilityof such molecules to cross membranes will of course enhance theirability to reach their target which is typically located inside of acell.

It will also be appreciated that the same means for facilitating orenhancing the transport of peptide inhibitors across cell membranes asdiscussed above are also applicable to the CD137 protein or its isoformsthemselves as well as other peptides and proteins which exert theireffects intracellularly.

As regards the antibodies mentioned herein throughout, the term“antibody” is meant to include polyclonal antibodies, monoclonalantibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id)antibodies to antibodies that can be labeled in soluble or bound form,as well as fragments thereof provided by any known technique, such as,but not limited to enzymatic cleavage, peptide synthesis or recombinanttechniques, and especially single-chain (sc) antibodies, which have theadvantage that they are coded for by a single chain of nucleic acids andmay therefore easily be introduced into and expressed in cells.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which populations containssubstantially similar epitope binding sites. MAbs may be obtained bymethods known to those skilled in the art. See, for example Kohler andMilstein, Nature, 256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubelet al., eds., Harlow and Lane ANTIBODIES: A LABORATORY MANUAL, ColdSpring Harbor Laboratory (1988); and Colligan et al., eds., CurrentProtocols in Immunology, Greene Publishing Assoc. and Wiley InterscienceN.Y., (1992-1996), the contents of which references are incorporatedentirely herein by reference. Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclassthereof. A hybridoma producing a mAb of the present invention may becultivated in vitro, in situ or in vivo. Production of high titers ofmAbs in vivo or in situ makes this the presently preferred method ofproduction.

Chimeric antibodies are molecules of which different portions arederived from different animal species, such as those having the variableregion derived from a murine mAb and a human immunoglobulin constantregion. Chimeric antibodies are primarily used to reduce immunogenicityin application and to increase yields in production, for example, wheremurine mAbs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric mAbs are used. Chimericantibodies and methods for their production are known in the art(Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984);Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984);Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., EuropeanPatent Application 125023 (published Nov. 14, 1984); Neuberger et al.,Nature 314:268-270 (1985); Taniguchi et al., European Patent Application171496 (published Feb. 19, 1985); Morrison et al., European PatentApplication 173494 (published Mar. 5, 1986); Neuberger et al., PCTApplication WO 8601533, (published Mar. 13, 1986); Kudo et al., EuropeanPatent Application 184187 (published Jun. 11, 1986); Sahagan et al., J.Immunol. 137:1066-1074 (1986); Robinson et al., International PatentApplication No. WO8702671 (published May 7, 1987); Liu et al., Proc.Natl. Acad. Sci USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad.Sci USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988);and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra. Thesereferences are entirely incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody, which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g. mouse strain) as the sourceof the mAb to which an anti-Id is being prepared. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody by producing an antibody to these idiotypicdeterminants (the anti-Id antibody). See, for example, U.S. Pat. No.4,699,880, which is herein entirely incorporated by reference.

Such anti idiotypic antibodies may be used to stimulate growth,proliferation, differentiation and/or activation of hematopoietic stemcells, just like CD137 as described further hereinabove.

The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

Accordingly, mAbs generated against the CD137 proteins, analogs,fragments or derivatives thereof, of the present invention may be usedto induce anti-Id antibodies in suitable animals, such as BALB/c mice.Spleen cells from such immunized mice are used to produce anti-Idhybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can becoupled to a carrier such as keyhole limpet hemocyanin (KLH) and used toimmunize additional BALB/c mice. Sera from these mice will containanti-anti-Id antibodies that have the binding properties of the originalmAb specific for an epitope of the above CD137 protein, or analogs,fragments and derivatives thereof.

The anti-Id mAbs thus have their own idiotypic epitopes, or “idiotopes”structurally similar to the epitope being evaluated, such as CD137Ligand extracellular epitopes.

The term “antibody” is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fab and F(ab′)2, whichare capable of binding antigen. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies useful in the present invention may be used for the detectionand quantitation of the CD137 protein according to the methods disclosedherein for intact antibody molecules. Such fragments are typicallyproduced by proteolytic cleavage, using enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab′)2 fragments).

As mentioned hereinabove, Fab or F(ab′)2 fragments, or antibodies orsingle chain antibodies or anticalins or Trinectins or the likemolecules, that specifically bind to CD137 ligand(s) as expressed uponthe cell surface of, e.g., activated T or B cells, or on the surface ofstem cells. Fragments may of course also be used as functional CD137analogs, so that when these molecules are multimerized, they may bindand cross-link CD137 ligands on target cells and thereby stimulateproliferation, growth, differentiation and/or activation of stem cells.

An antibody is said to be “capable of binding” a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

The antibody of the invention may not only bind the CD137 protein, butalso inhibit its biological activity. This may be tested easily bymethods known to the person of skill in the art. For instance, addingvarious amounts of the antibody to cells bearing CD137 or to a solutioncontaining crosslinked CD137 protein will result in a lower stimulatingactivity observed if that antibody is capable of inhibiting thebiological activity of CD137, as compared to irrelevant controlantibody. Thus, antibodies can be identified that specifically inhibitCD137 activity. Such antibodies are useful in control assays for CD137activity. Such antibodies are also useful in inhibiting endogenous orexogenous CD137 activity in mammals, tissues, or cells, preferably stemcells, more preferably hematopoietic stem cells, including peripheraland bone marrow stem cells, most preferably on bone marrow stem cells.

The CD137 proteins of the invention may be produced by any standardrecombinant DNA procedure (see for example, Sambrook, et al., 1989 andAusubel et al., 1987-1995, supra) in which suitable eukaryotic orprokaryotic host cells well known in the art are transformed byappropriate eukaryotic or prokaryotic vectors containing the sequencesencoding for the proteins. Accordingly, the present invention alsoconcerns such expression vectors and transformed hosts for theproduction of the proteins of the invention. As mentioned above, theseproteins also include their biologically active analogs, fragments andderivatives, and thus the vectors encoding them also include vectorsencoding analogs and fragments of these proteins, and the transformedhosts include those producing such analogs and fragments. Thederivatives of these proteins, produced by the transformed hosts, arethe derivatives produced by standard modification of the proteins ortheir analogs or fragments.

The invention is now more fully described and illustrated by theexamples as set out below. It is understood that the examples are notlimiting the scope of the invention, and that e.g. other receptors ormolecules or gene sequences may be cloned according to the teaching ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of human CD137 on proliferation of peripheralstem cells,

FIG. 2 a shows IL-6 secretion induced in murine monocytes by humanCD137,

FIG. 2 b shows the effects of human CD137 on murine stem cells,

FIG. 3 shows a comparison of CD137- and G-CSF-induced stem cellproliferation,

FIG. 4 shows CD137 effects on the different hematopoietic lineages.RFUs: relative fluorescent units.

FIG. 5 shows CD137 effects on the different hematopoietic lineages inthe absence of other growth factors. RFUs: relative fluorescent units

FIG. 6 shows CD137 effects on the myeloid lineage under different growthfactor concentrations. RFUs: relative fluorescent units.

FIG. 7 shows a comparison of CD137-induced proliferation anddifferentiation of the myeloid lineage. RLUs: Relative fluorescentunits, RLUs: Relative luminescent units.

FIG. 8 shows a comparison of proliferation of human hematopoietic stemcells brought into contact with COS cells that were either untreated(left), expressing empty vector (middle), or expressing human CD137(right bar).

Tables 2-4 show data relating to a reconstitution of the hematopoieticsystem by CD137 in vivo.

EXAMPLES Example 1 Effect of Human CD137 on Peripheral Stem Cells

Human hematopoietic stem cells were isolated from the bone marrow ofhealthy donors via their CD34 expression by positive selection using the“Direct CD34 Progenitor Kit” (Miltenyi, Bergisch Gladbach, Germany). 96well plates were coated with 1 μg/ml of CD137-Fc protein or an equimolaramount of Fc protein (0.5 μg/ml). 10⁵ cells were plated per well andincubated for 3, 5 or 8 days. During the last 12 h the cells werelabelled with 0.5 μCi ³H-thymidine. The rate of proliferation wasdetermined with a szintillation counter (Packard, Meriden, Conn.). Eachcondition was done in triplicates.

FIG. 1 shows that CD137 is able to induce proliferation of humanhematopoietic stem cells. Compared to the Fc control proteins, CD137-Fcprotein induces a 8-20 fold increase in DNA synthesis, as evidenced byincorporation of ³H-thymidine. This activity of CD137 is long-lasting,as it is evident at day three, five and eight.

Example 2 Effects of Human CD137 on Murine Stem Cells

In order to be able to test the effects of human CD137 on murinehematopoietic stem cells, crossreactivity of human CD137 with the murineCD137 Ligand was verified. Human CD137 (h CD137 Fc) induced IL-6secretion in murine monocytes in the same way as it did in humanmonocytes (see FIG. 2A for induction by human CD137 Fc on IL-6 secretionof murine monocytes). Also murine CD137-Fc was able to induce IL-6production in murine monocytes (FIG. 2A, m CD137-Fc). The experimentshown in FIG. 2 a was carried out by isolating murine peritoneal exudatecells (>90% monocytes) from the peritoneum of BALB/C mice. 96 wellplates were coated with 1 mg/ml of human (h CD137-Fc) or murine CD137-Fc(mCD137-Fc) protein or an equimolar amount of human Fc protein (Fc, 0.5mg/ml). 10⁵ cells were plated per well and incubated for 16 h.Concentrations of IL-6 in supernatantes were determined by ELISA. Eachcondition was done in triplicates.

In another experiment, bone marrow cells were isolated from the femurbones of adult NMRI mice. Both ends of the bones were cut and using asyringe the marrow was flushed out with PBS. Tissue particles wereremoved by a fine-meshed sieve. Flow through cells were washed with PBS.96 well plates were coated with 1 μg/ml CD137-Fc (CD137 immob) proteinor an equimolar amount of Fc (Fc Immob) protein (0.5 μg/ml).Alternatively, both proteins were added as soluble forms (sCD137 andsFc). 5×10⁴ cells were plated per well and cultured for 7 days. Cellswere labelled with 0.5 μCi ³H-thymidine during the last 12 h of theexperiment. The rate of proliferation was determined with aszintillation counter (Packard, Meriden, Conn.). Each condition was donein triplicates.

FIG. 2 b shows that proliferation is induced in murine hematopoieticstem cells (MHSC) isolated from the marrow of the femur bone by humanCD137. The rate of proliferation induced is similar to the values seenin FIG. 1. Culture of MHSC on immobilized CD137-Fc protein (CD137immob.) increased cell proliferation about 10-fold compared toimmobilised Fc control protein. In its soluble form, CD137-Fc (sCD137)had no effect on cell proliferation, and neither did soluble Fc controlprotein (sFc).

Example 3 Comparison of CD137 and G-CSF Induced Stem Cell Proliferation

So far Neupogen (G-CSF) is being used to reconstitute the hematopoieticsystem after chemo- or radiation therapy. Therefore, it was interestingto compare the effects of CD137 and G-CSF on the proliferation ofhematopoietic stem cells.

G-CSF is fast acting but its effects last only a few hours (max. 30 h).Due to its short half-life G-CSF is generally administered daily or evenseveral times a day in animal models (Okabe et al., 1990; Aso and Akaza,1992).

CD137 has a significantly slower kinetics with a slower onset ofactivity but also a prolonged period of activity. G-CSF was added dailyfor the first four days whereas CD137 was only given once on the firstday of the experiment.

Bone marrow cells were isolated from the femur bones of adult NMRI mice.Both ends of the bones were cut and using a syringe the marrow wasflushed out with PBS. Tissue particles were removed by a fine-meshedsieve. Flow through cells were washed with PBS. 96 well plates werecoated on day 0 with 50 μg/well of 1.2 μg/ml CD137-Fc protein (=60 ng,CD137-Fc imm.) or an equimolar amount of Fc protein (50 ml of 0.6 μg/ml,Fc imm.). Alternatively, 60 ng of CD137-Fc (Pr. A CD137-Fc) or 30 ng Fcprotein (Pr. A Fc) immobilized on protein A by incubation at RT for 1 hwere added. Soluble protein A was used, rather that protein A coupled tobeads. CD137-Fc and Fc, either immobilized on the plate or immobilizedon soluble protein A were added solely on day 1. Human G-CSF (R&DSystems) was added daily from day 1 to 4 at a concentration of 200 ng/ml(G-CSF). 5×10⁴ cells were added per well and cultured for 3, 5 or 7days. During the last 4 h the cells were labelled with MTS(3-(4,5-dimethydiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sufophenyl)-2H-tetrazolium)+PES(phenazine ethosulfat) obtained from Promega. The rate of proliferationwas determined at OD₄₉₀. Each condition was done in triplicates.

CD137-Fc immobilized on the tissue culture plate had the strongesteffect at day 3, 5 and 7 and more than doubled proliferation of thecells (FIG. 3, CD137-Fc imm.). The values for day 7 are an underestimatebecause the wells with immobilized CD137 were already overgrown by thattime and cell growth was subject to contact inhibition.

CD137-Fc, which was immobilized on protein A (FIG. 3, Pr. A CD137-Fc)had about ⅔ of the activity of plate-immobilized CD137-Fc and was aseffective as G-CSF (FIG. 3, G-CSF). 1×60 ng CD137-Fc were as or evenmore active than 4×20 ng G-CSF. The controls, plate-immobilized Fc (Fcimm.) and protein A-immobilized Fc (Pr. A Fc) and soluble Fc wereinactive (FIG. 3).

Example 4 Evaluation of CD137 Effects on the Different HematopoieticLineages

The effects of CD137 on the different hematopoietic lineages wereevaluated using a cell based immunoassay, which quantifies expression oflineage-specific markers (CELISA, BioWhittaker, Walkersville, Md., USA).

Progenitors for the myeloid and erythroid lineages were normal humanCD34+ cells, which had been isolated from the bone marrow of healthyvolunteers by positive selection using the “Direct CD34 Progenitor Kit”(Miltenyi, Bergisch Gladbach, Germany). Progenitors for themegakaryocyte lineage were normal human CD34+ cells, which had beenisolated from the peripheral blood of healthy G-CSF-treated volunteers.

96-well polystyrene plates were precoated with four dilutions ofCD137-Fc or Fc control protein at 4° C. overnight or with the dilutionbuffer PBS. The following additional controls were included in theexperiment: no addition; Camptothecin, a cytotoxic compound, at 0.005 μM(condition 1) and at 0.5 μM (condition 2); stem Cell Factor (SCF) at aconcentration of 125 ng/ml. The concentrations of the agents in thedifferent conditions shown in FIG. 4 were thus:

-   Condition 1: (in the Figure, bars indicated with “1”, from left to    right) PBS; CD137-Fc 1.2 mg/ml; Fc 0.6 mg/ml; “---”: no addition;    Camptothecin 0.005 mM; SCF 125 ng/ml;-   Condition 2: (in the Figure, bars indicated with “2”, from left to    right) CD137-Fc 0.6 mg/ml; Fc 0.3 mg/ml; Camptothecin 0.5 mM;-   Condition 3: (in the Figure, bars indicated with “3”, from left to    right) CD137-Fc 0.3 mg/ml; Fc 0.15 mg/ml;-   Condition 4: (in the Figure, bars indicated with “4”, from left to    right) CD137-Fc 0.15 mg/ml; Fc 0.075 mg/ml.

A volume of 50 μl was used per well. All measurements were done intriplicate. Plates were washed and 2×10³ human CD34+ progenitors wereadded per well. Cells were cultured in standard cell culture medium(RPMI, 15% fetal calf serum, PenStrep, L-Gln). Lineage specificdifferentiation to the myeloid, erythroid and megakaryocyte lineage wasinduced by addition of the factors listed in Table 1. TABLE 1 Table 1:Lineage specific differentiation was induced in human CD34+ progenitorcells by addition of the listed factors. Reagent Myeloid ErythroidMegakaryocyte SCF  25 ng/ml 25 ng/ml 25 ng/ml GM-CSF  1 ng/ml none  1ng/ml G-CSF  1 ng/ml none none Transferrin 200 ug/ml none none EPO None 3 U/ml none TPO None none 10 ng/ml

PBS, no addition (=---) and SCF were only included in condition 1.Camptothecin was only included in condition 1 and 2 (at differentconcentrations, as described above). Conditions 1, 2, 3 and 4 only referto different concentrations of CD137 and Fc.

After 10 days of incubation, the cells were transferred to ELISA plates(with 3× multiple washes) for the measurement of expression oflineage-specific cell surface markers. CD11b was used for the myeloidlineage, CD41a (platlet glycoprotein IIb/IIIa) was used for themegakaryocyte lineage and glycophorin A was used for the erythroidlineage.

Results:

The two controls “PBS” and “no-addition” controls gave similar resultsin all three lineages, indicating that PBS, which was used as a solventfor Fc and CD137-Fc protein had no effect on cell growth.

As expected, the negative control, 0.005 uM Camptothecin showed minimalgrowth inhibition and at 0.05 uM Camptothecin inhibited growth to agreater extent in all lineages.

The CD137 Fc protein increased proliferation of the myeloid precursorcells at all four concentrations (0.6-0.075 μg/ml).

The CD137-Fc protein had no effect on the growth of erythroidprecursors. Both, the CD137-Fc and the Fc control protein reduced growthof the megakaryocytic lineage, implying a toxic effect of the Fc domain.CD137 may have partly counteracted that inhibitory effect.

Example 5 Evaluation of CD137 Effects on the Different HematopoieticLineages in the Absence of Other Growth Factors

Example 4 had established that CD137 induces proliferation ofhematopoietic precursor cells, in particular that of the myeloid andmegakaryocyte lineages.

In the experiment depicted in FIG. 4, optimal amounts of growth factorswere present (see Table 1 above). These growth factors may be essentialcofactors for CD137 activity but their activity may also mask that ofCD137. Therefore, growth factors were omitted in Example 5. The cellculture medium contained only 15% fetal calf serum, PenStrep and L-Gln.Otherwise, the experimental set up was identical to that in Example 4.

The concentrations of the agents in the different conditions shown inFIG. 5 were thus:

-   Condition 1: (in the Figure, bars indicated with “1”, from left to    right) Fc 0.6 mg/ml; CD137-Fc 1.2 mg/ml; SCF 125 ng/ml; “---”: no    addition.-   Condition 2: (in the Figure, bars indicated with “2”, from left to    right) Fc 0.3 mg/ml; CD137-Fc 0.6 mg/ml;-   Condition 3: (in the Figure, bars indicated with “3”, from left to    right) Fc 0.15 mg/ml; CD137-Fc 0.3 mg/ml.-   Condition 4: (in the Figure, bars indicated with “4”, from left to    right) Fc 0.075 mg/ml; CD137-Fc 0.15 mg/ml.

As expected, proliferation of the all three lineages was markedlyreduced in the absence of growth factors. CD137 enhanced growth of themyeloid lineage and the megakaryocyte lineage as it has done in thepresence of growth factors. In contrast to Example 4, no inhibitoryeffect of the Fc domain on the megakaryocyte precursors was noticeable.But whereas CD137 had no effect on the growth of the erythroid lineagein the presence of growth factors, it significantly enhanced erythroidproliferation and/or differentiation in the absence of other growthfactors. This suggests that the presence of growth factors in Example 4had masked the activating effect of CD137 on erythroid cells.

These data indicate that CD137 is sufficient for induction of growth andproliferation of the myeloid, megakaryocyte, and erythroid lineage.

Example 6 Growth Factor Dependency of CD137 Effects on the MyeloidLineage

Examples 4 and 5 show that CD137 induces proliferation of myeloidprecursor cells, in the presence as well as in the absence of othergrowth factors. In this example, the influence of growth factorconcentrations and potential additive or synergistic effects of thesegrowth factors with CD137 were analysed in more detail. Growth factorconcentrations were reduced to 50%, 5% and 0% compared to example 4.Further, only myeloid precursor cells were used. Otherwise theexperimental set up was identical to that of the myeloid lineage inexample 4.

PBS and Fc served as negative controls. The concentrations of the agentsin the different conditions shown in FIG. 6 were:

-   Condition 1: (in the Figure, bars indicated with “1”, from left to    right) Fc 0.6 mg/ml; CD137-Fc 1.2 mg/ml. SCF 125 ng/ml.-   Condition 2: (in the Figure, bars indicated with “2”, from left to    right) Fc 0.3 mg/ml; CD137-Fc 0.6 mg/ml.-   Condition 3: (in the Figure, bars indicated with “3”, from left to    right) Fc 0.15 mg/ml; CD137-Fc 0.3 mg/ml.-   Condition 4: (in the Figure, bars indicated with “4”, from left to    right) Fc 0.075 mg/ml; CD137-Fc 0.15 mg/ml.-   GF: growth factors as in Table 1.

CD137-Fc enhanced proliferation most significantly when other myeloidgrowth factors were present (FIG. 6, upper panel). At 50% growth factorconcentrations, CD137-Fc was as or even more active as the positivecontrol, stem cell factor (SCF). The same pattern was observed with theother positive control, the 100% growth factor condition (100% GF,4^(th) bar from the left in condition 1). CD137-Fc not only compensatedfor the missing 50% growth factors but increased proliferation beyondthat obtained with the 100% growth factor control.

Reduction of growth factor concentrations did not result in a strongerinduction of growth by CD137. In contrast, the stimulating CD137 effectson myeloid precursors were less prominent at 5% and 0% growth factorconcentrations (FIG. 6, middle and lower panel, respectively). Thesedata suggest that although as demonstrated by Example 5 CD137 can inducegrowth on its own, it does work in combination with one or several ofthe growth factors listed in Table 1.

Example 7 Comparison of CD137-Induced Proliferation and Differentiation

Differentiation was assessed per CELISA (BioWhittaker), measuring theexpression of the myeloid-specific protein CD11b. Proliferation wasdetermined using the ViaLight system (BioWhittaker), which measures theamount of ATP in the culture. Cell culture conditions were as describedin Example 6. Only myeloid precursors were used.

The concentrations of the agents in the different conditions shown inFIG. 7 were:

-   Condition 1: (in the Figure, bars indicated with “1”, from left to    right) Fc 0.6 mg/ml; CD137-Fc 1.2 mg/ml; SCF 125 ng/ml.-   Condition 2: (in the Figure, bars indicated with “2”, from left to    right) Fc 0.3 mg/ml; CD137-Fc 0.6 mg/ml.-   Condition 3: (in the Figure, bars indicated with “3”, from left to    right) Fc 0.15 mg/ml; CD137-Fc 0.3 mg/ml.-   Condition 4: (in the Figure, bars indicated with “4”, from left to    right) Fc 0.075 mg/ml; CD137-Fc 0.15 mg/ml.

At 50% growth factor concentrations (see Table 1 for the list andconcentrations of growth factors) the absolute amounts of proliferationand differentiation were larger than at 5%, confirming the results ofExample 6, that CD137 works in combination with other growth factors.

At 5% growth factor concentrations the CD137-induced increase inproliferation compared to the Fc control protein was larger thanCD137-induced increase in differentiation. Proliferation was increasedby CD137 about threefold (from 2.000 to around 6.000 RLUs; FIG. 7,bottom left panel), whereas differentiation was increased about twofold(from 25.000 to around 50.000 RLUs; FIG. 7, top left panel).

The reverse pattern was seen at 50% growth factor concentrations wherethe relative increase in proliferation was smaller, while the relativeincrease in differentiation was larger. The comparison is again based onvalues obtained with the Fc control protein. At 50% growth factorconcentrations proliferation was increased by CD137 less than twofold(from 7.000 to 8.000-13.0000 RLUs, depending on the CD137-Fcconcentration; FIG. 7, bottom right), whereas differentiation wasincreased about 7-fold (from 50.000 to around 350.000 RLUs; FIG. 7, topright).

This implies that the rate of differentiation of hematopoietic stemcells increases with increasing growth factor concentrations and thusthat differentiation is mainly mediated by the added growth factors.Proliferation on the other hand can be induced by CD137 without much orany contribution from the growth factors. This finding is consistentwith data from Example 1-3 where CD137-induced proliferation in theabsence of other growth factors.

Nevertheless, CD137 can also contribute significantly todifferentiation. The 100% growth factor control induced an about 10-foldhigher degree of differentiation compared to 5% growth factorconcentrations (200.000 RFUs vs 20.000 in the PBS control; FIG. 7, topleft panel). The combination of CD137-Fc and 5% growth factors alsoresulted in a lower degree of differentiation (50.0000 RFU; FIG. 7, topleft panel). However, at 50% of growth factor concentrations CD137 notonly compensated for the remaining 50% growth factors but increaseddifferentiation markedly is beyond that level (350.000 vs 200.000 RFUs;FIG. 7, top right panel)

Though CD137-induced proliferation of myeloid precursor cells is notdependent on other growth factors they clearly support it. This isevidenced by the higher proliferation rates at 50% compared to 5% growthfactor concentrations (8.000-13.0000 vs 5.000 RLUs, FIG. 7, bottom rightvs bottom left panel).

Example 8 Effect of CD137-Transfected Cells on Hematopoietic Stem Cells

Human hematopoietic stem cells were isolated from the bone marrow ofhealthy donors via their CD34 expression by positive selection using the“Direct CD34 Progenitor Kit” (Miltenyi, Bergisch Gladbach, Germany).

COS cells were transfected with a CD137 expression vector (pCD137,containing the entire human CD137 cDNA coding sequence) or the emptyvector (pcDNA3), respectively. Untransfected COS cells were used as anadditional control. 3×10³ COS cells were seeded into wells of a 96 wellplate and grown for 2 days to confluency. Cells were fixed with 0.25%glutaraldehyde for 10 min at room temperature and were then washed twicewith PBS. 5×10⁴ human hematopoietic stem cells (HSC) were plated perwell and cultured for 7 days. HSC were isolated from the peripheralblood of healthy volunteers via CD34 expression. Cells were labelledwith 0.5 μCi ³H-thymidine during the last 12 h of the experiment. Therate of proliferation was determined with a szintillation counter(Packard, Meriden, Conn.). Each condition was done in triplicates.

FIG. 8 shows that CD137 when expressed ectopically on transfected cellsis able to induce proliferation of human hematopoietic stem cells.Compared to untransfected (untransfected) and vector-transfected(pcDNA3) COS cells, CD137-transfected cells (pCD137) induce a more than5-fold increase in DNA synthesis, as evidenced by incorporation of³H-thymidine. This activity of CD137 is long-lasting, as it is evidentat day seven.

Example 9 In vivo Efficacy of CD137 in Reconstituting the HematopoieticSystem

CD137 needs to be immobilized onto a carrier or crosslinked to befunctional according to the invention. Protein A was chosen as a carrierfor in vivo studies, as it has been effective in crosslinking CD137-Fcin the experiment described in Example 3. CD137-Fc or Fc control proteinwere immobilized on protein A and injected into NMRI mice. Besidesimmobilizing CD137-Fe or Fc, protein A has the additional effect ofbeing toxic for cells of the hematopoietic system. This latter activityof protein A exerts a damaging effect on the hematopoietic system ofmice as does chemotherapy.

Three mice per group were used. Each mouse received 100 μg ofimmobilized CD137-Fc or an equimolar amount of immobilized Fc or proteinA alone. Mice were killed after 3 weeks and the bone marrow was isolatedfrom the femurs and analysed (Table 2). TABLE 2 Table 2: Bone marrowcomposition on day 21. Protein Protein A Cell types untreated Protein AA-Fc CD137-Fc R1 91% erythroid 56.15 69.18 74.51 55.67 R2 87% lymphoid18.21 25.35 21.95 18.54 R3 49% undifferentiated blasts 0.73 1.81 1.070.87 25% lymphoblasts 18% basophil erythroblasts R4 50% undifferentiatedblasts 5.50 1.97 1.06 4.92 30% myeloid mainly und. precursors 20%erythroid R5 91% granulocytes 14.21 1.12 1.33 16.17 R6 74% monocytes5.20 0.63 0.13 3.83 20% myeloid precursorsPercentage of total cells.Average, of three mice.

The relative composition of the bone marrow cell population was severelydisturbed by the protein A treatment. Crossinking of the Fc controlprotein onto protein A had no effect on the damaging activity of proteinA. The numbers of granulocytes and monocytes were reduced drastically inthe protein A (Pr.A) as well as in the Pr.A-Fc mice, demonstrating themyelotoxic effect of protein A. The percentages of these cells in thePr.A-CD137-Fc mice were far higher (16.17 vs 1.33 and 3.83 vs 0.13%,respectively), and comparable to that of the untreated mice. These dataindicate that CD137 induces proliferation of hematopoietic stem cellsalso in vivo and that it is able to reverse damage to the hematopoieticsystem. Also, a single application of immobilized CD137 is sufficientfor restoration of the hematopoietic system.

In the next experiment NMRI mice were treated with Alkeran, a cytotoxicdrug regularly used for human cancer therapy. Alkeran was given i.p. ondays 1, 3 and 5. Mice received 100 μg of CD137-Fc immobilized on proteinA or equivalent amounts of immobilized Fc control protein. Three micewere used per group. The relative composition of the bone marrow cells(Table 3) and the absolute number of cells in the peripheral blood(Table 4) were determined after 21 days. TABLE 3 Table 3: Bone marrowcomposition on day 21. Un- Alkeran Alk. + Pr. Alk + Pr. A- Cell typestreated (Alk.) Alk. + Pr. A A-Fc CD137-Fc R1 51.24 60.21 77.36 71.1852.20 R2 18.06 22.54 19.12 15.93 19.88 R3 0.66 0.66 0.69 0.63 0.64 R45.87 2.28 1.85 3.19 3.94 R5 19.53 11.56 0.51 8.05 18.04 R6 4.87 2.240.80 1.29 5.59Percentage of total cells.Average of three mice.Please see Table 2 for key to cell types (R1-R6).

TABLE 4 Table 4: Total number of leukocytes, erythrocytes andthrombocytes in the peripheral blood per μl. Un- Alkeran Alk. + Pr.Alk + Pr. A- Cell types treated (Alk.) Alk. + Pr. A A-Fc CD137-FcLeukoc. 1.600 830 880 680 1.410 Erythroc 3.910 3.820 3.680 3.870 3.880Thrombo 652.000 581.000 573.000 596.000 583.000Average of three mice.

Alkeran significantly reduced the percentage of granulocytes (R5) andmonocytes (R6) and the toxic effect of protein A-Fc reduced them evenfurther. Again, a single dose of CD137 restored the hematopoieticsystem. Its therapeutic effect even outlasted two additional doses ofAlkeran, given 2 and 4 days after CD137.

In this experiment not only relative but also absolute cell numbers weredetermined. This excludes the possibility that the increase in thepercentage of monocytes or granulocytes may be due to a decrease inother cell types. These in vivo results confirm data in Examples 1 to 8,which show induction of hematopoietic stem cell proliferation by CD137in vitro.

REFERENCES

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1. CD137 or a functional analogue thereof, for use in induction ofproliferation of hematopoietic stem cells of a mammal.
 2. CD137 or afunctional analogue thereof according to claim 1, for use in inductionof proliferation of monocytic precursor cells.
 3. CD137 or a functionalanalogue thereof according to claim 1, for use in stimulatinghematopoiesis.
 4. CD137 or a functional analogue thereof according toclaim 1, for use in tissue repair, tissue regeneration and woundhealing.
 5. CD137 or a functional analogue thereof according to claim 1,for use in enhancing innate and/or adaptive immunity for cancer therapy.6. CD137 or a functional analogue thereof according to claim 1, for usein enhancing innate and/or adaptive immunity for therapy of infectiousdisease.
 7. CD137 or a functional analogue thereof according to claim 1,for use in enhancing innate and/or adaptive immunity for vaccinationagainst infectious disease.
 8. A method of treatment of a disordercharacterized by insufficient numbers of cells of the hematopoieticsystem, including but not limited to T cells, B cells, granulocytes,macrophages, mesenchymal cells, osteoclasts and multipotent adultprogenitor cells, comprising the step of administration to a mammal inneed thereof of an effective dose of CD137 or a functional analoguethereof.
 9. A method of treatment for a disorder characterized by aninsufficient number of cells of the hematopoietic system, including butnot limited to T cells, B cells, granulocytes, macrophages, mesenchymalcells, osteoclasts and multipotent adult progenitor cells comprising thestep of administration of CD137 or a functional analogue thereof to anisolated culture of stem cells and the transfer of the treated cells toa mammal in need thereof.
 10. A method for the treatment according toclaim 8 of a mammal in need thereof wherein the mammal is characterizedby having a decrease in the number or activity of its white blood cells.11. A method according to claim 10, wherein the decrease is caused by orassociated with an immunodeficiency.
 12. A method according to claim 11,wherein the immunodeficiency is selected from the group comprising AIDS,hyperimmunoglobulin M syndrome, radiation-induced immunodeficiency,chemotherapy-induced immunodeficiency.
 13. A method according to claim10, wherein the decrease is associated with chemo- and/or radiotherapyand/or removal of blood progenitor cells.
 14. A method according toclaim 13, wherein the chemo- and/or radiotherapy and/or removal of bloodprogenitor cells is administered to treat cancer.
 15. A method accordingto claim 13, wherein the chemo- and/or radiotherapy and/or removal ofblood progenitor cells is administered to treat autoimmune disease. 16.A method of claim 10, wherein the decrease is associated withleukopenia.
 17. A method of claim 16, wherein the leukopenia is causedby a condition selected from the group comprising severe trauma, bloodloss, immunodeficiency, or disease such as agranulocytosis or bonemarrow failure, wherein said Bone marrow failure may for instance be dueto congenital factors, toxins such as benzene, street drugs, viralinfections such as hepatitis, or side effects of immunotherapies.
 18. Amethod of claim 16, wherein the leukopenia is caused by a disease suchas anemia, aplastic anemia, megablastic anemia, myelophthisic anemia,myelodysplastic syndrome or hairy cell leukemia.
 19. A method accordingto claim 8, wherein the dose of CD137 protein administered is within therange of 1 ng/kg to 1 mg/kg, more preferably 50 ng/kg to 500 μg/kg. 20.A method according to claim 19, wherein the dose of CD137 proteinadministered is within the range 100 ng/kg to 100 μg/kg more preferably500 ng/kg to 50 μg/kg.
 21. A method according to claim 20, wherein thedose of CD137 protein administered is within the range of 1 to 10 μg/kgmore preferably 4 to 6 μg/kg.
 22. A method for the stimulation ofgrowth, proliferation, differentiation and/or activation ofhematopoietic stem cells, comprising the step of contacting the cellswith an effective amount of CD137, during a time period sufficient toallow for said the stimulation of growth, proliferation, differentiationand/or activation.
 23. A method of claim 22, wherein the concentrationof CD137 is from about 1 ng/ml to about 1 mg/ml.
 24. A method of claim23, wherein the concentration of CD137 is from about 5 ng/ml to about400 mg/ml.
 25. A method of claim 24, wherein the concentration of CD137is from about 15 ng/ml to about 100 ng/ml.
 26. A method of claim 25wherein the concentration of CD137 is about 60 ng/ml.
 27. A method ofclaim 8, wherein the CD137 or functional analogue thereof is CD137, or apart of CD137, fused to Fc.
 28. A method of claim 27 wherein the CD137contains the extracellular part thereof.
 29. A method of claim 28wherein the CD137 contains amino acids 18 to 255 thereof.
 30. A methodof claim 29 wherein the CD137 contains amino acids 18 to 186 thereof.31. A method of claim 8 wherein the CD137 or functional analogue thereofis used in combination with a growth factor.
 32. The method of claim 31wherein the growth factor is selected from among G-CSF, M-CSF, GM-CSF,IL-3, IFN-gamma, TNF, LIF, flt-3, c-kit.
 33. The method of claim 32wherein the growth factor is selected from among G-CSF, M-CSF andGM-CSF.
 34. The method of claim 33 wherein the growth factor is G-CSF.35. The method of claim 8 wherein the CD137 or functional analoguethereof is administered as a single dose.
 36. A CD137 molecule, orfunctional analogue thereof, which is multimerized, for use in a methodof claim
 8. 37. The molecule of claim 36, wherein the multimer comprises2 to 20 monomers.
 38. The molecule of claim 37, wherein the multimercomprises 3 to 10 monomers.
 39. The molecule of claim 37, wherein themonomers are expressed a fusion protein.
 40. The molecule of claim 37wherein the monomers are fused together by means of a covalent bond. 41.A molecule capable of crosslinking CD137 ligand(s) expressed on thesurface of a target cell, for use according to claims
 1. 42. Themolecule of claim 41 which is an antibody or derived from an antibody.43. The molecule of claim 41 which is an anticalin or derived from ananticalin.
 44. The molecule of claim 41 which is a Trinectin or derivedfrom a Trinectin.
 45. The molecule of claim 36, characterized by theability to stimulate the growth, proliferation, differentiation and/oractivation of stem cells.
 46. The molecule of claim 45, wherein the stemcells are hematopoietic stem cells.
 47. The molecule of claim 45,wherein the stem cells are CD34 positive cells.
 48. A compositioncomprising CD137 or a functional analogue thereof, and a diluent and/orcarrier.
 49. The composition of claim 48, for dermal, transdermal, oral,intravenous, intraperitoneal, intramuscular, or intraliquorealadministration.
 50. The composition of claim 48, for use in thetreatment of a disorder of a mammal wherein the disorder is associatedwith decreased number or activity of white blood cells.
 51. Thecomposition of claim 50, wherein the decrease is caused by or associatedwith an immunodeficiency.
 52. The composition of claim 51, wherein theimmunodeficiency is selected from the group comprising AIDS,hyperimmunoglobulin M syndrome, radiation-induced immunodeficiency,chemotherapy-induced immunodeficiency.
 53. A composition of claim 48 foruse in stimulation of proliferation and/or differentiation ofhematopoietic stem cells as may be beneficial in disorders such asimmunodeficiency, agranulocytosis, bone marrow failure, anemia, aplasticanemia, megablastic anemia, myelophthisic anemia, myelodysplasticsyndrome or hairy cell leukemia.
 54. A method of treatment of a mammalwherein a therapeutically effective amount of a composition of claim 48is administered to a mammal in need thereof, the mammal having adisorder associated with decreased proliferation and/or differentiationof stem cells.
 55. The method of claim 54 wherein the disorder is cancerof cells or the hematopoietic lineage.
 56. The method or composition ofclaim 55, wherein the disorder is leukemia.
 57. CD137 or a functionalanalogue thereof, for use in induction of proliferation of stem cells ofa mammal.
 58. A method for the treatment of a mammal suffering from adisorder or condition selected from the group consisting ofimmunodeficiency, agranulocytosis, bone marrow failure, anemia, aplasticanemia, megablastic anemia, myelophthisic anemia, myelodysplasticsyndrome and hairy cell leukemia, comprising administering an effectiveamount of a composition in accordance with claim
 57. 59. A moleculecapable of crosslinking CD137 ligand(s) expressed on the surface of atarget cell, for use in a method of claim
 8. 60. The composition ofclaim 48 wherein the disorder is cancer of cells of the hematopoieticlineage.